17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology - Book of Abstracts

Significance of the topic

Hyphenated techniques in chromatography and separation science combine complementary separation and detection technologies to extend analytical specificity, sensitivity and throughput. They are central to solving current challenges across pharmaceuticals (small molecules, oligonucleotides, proteins, antibody-drug conjugates), environmental monitoring (persistent organic pollutants, sediment cores, mineral oil hydrocarbons), food and flavor analysis, metabolomics/breathomics and forensic science. The HTC‑17 symposium (Ghent, May 2022) showcased advances that reduce time‑to‑result, improve molecular identification, enable analysis of highly complex or low‑volume matrices, and support regulatory and quality control demands in industry and academia.

Objectives and overview

The meeting assembled plenaries, keynotes, tutorials and short courses to: present state‑of‑the‑art hyphenated methods (GC×GC‑TOFMS, UHPLC‑HRMS, CE‑MS, SFC‑MS, SEC‑MS, AF4, TIMS, MALDI‑IMS), demonstrate practical method transfer and automation strategies, discuss sample‑preparation solutions (MIPs/IIPs, automated DBS, thermal desorption, SPME), and highlight informatics (retention prediction, data mining, AI). Special topics included high‑throughput/ultra‑fast GC, multidimensional LC (including HILIC×RPLC and RPLC×RPLC), large‑molecule hyphenation, proteoform analysis, and clinical/translational applications (breathomics, COVID‑19 diagnostics). A Journal of Chromatography A special issue invitation and multiple vendor seminars reinforced rapid dissemination and instrument translation.

Methodology and approaches

The program emphasized practical hyphenation strategies and key experimental variables:

• Comprehensive GC×GC‑TOFMS and TD‑GC×GC for volatile/semi‑volatile profiling, breathomics and fragrance analysis.
• Thermal‑gradient and flow‑field ultra‑fast GC methods to increase throughput (including low‑pressure GC / vacuum GC approaches).
• UHPLC‑HRMS, multidimensional LC (LC×LC, HILIC×RPLC, RPLC×RPLC) and middle‑up HILIC‑HRMS for glycoform and biopharmaceutical profiling.
• SFC‑MS with optimized make‑up solvent strategies and interfaces for chiral and achiral separations using CO2‑based mobile phases.
• Capillary electrophoresis‑MS (CE‑MS) and nanoLC‑CZE‑MS platforms for volume‑restricted metabolomics and intact/glycoprotein characterization.
• Native/affinity CE‑MS, SEC‑MS and AF4 hyphenations for proteoform‑resolved binding assays and aggregation/oligomer studies.
• Ion mobility (TIMS) and high‑resolution MS (Q‑Orbitrap, TOF) for additional separation dimension and confident identification.
• Sample preparation advances: molecularly/ion‑imprinted polymers (MIP/IIP) for selective extraction, automated DBS extraction, 3D‑printed sorbents and devices, and on‑line LC–GC coupling for MOSH/MOAH.
• Data science tools: QSRR and desirability indices for in‑silico method development, graph neural networks for retention prediction, automated data reduction for GC×GC, and workflows for non‑targeted studies.

Instrumentation used

The meeting illustrated broad instrument use and vendor developments: GC×GC TOFMS, GC‑APCI‑TIMS‑TOF, TD‑GC×GC‑FID/TOFMS, thermal desorption units, fast GC systems (HyperChrom, thermal gradient), UHPLC coupled to high‑resolution MS (Q‑Orbitrap, QTOF), SCIEX and Agilent MS platforms, CE‑MS interfaces, nanoLC and microfluidic/nanoflow LC devices, AF4 and SEC hyphenated to MS, MALDI‑IMS imaging hardware, SFC systems with various make‑up pump/interface designs, and multi‑detector UHPLC (CAD, single quadrupole MS). Additive manufacturing (3D printing) and specialized loop/modulator hardware for 2D separations were also highlighted.

Main results and discussion

• Biomarker and clinical applications: GC×GC‑TOFMS and TD‑GC×GC breathomics/blood volatilomics demonstrated discriminating markers for diseases (e.g., lung disease, IBD, COVID‑related studies) but emphasized the need for robustness, standardization and inter‑laboratory QA/QC for clinical translation.
• HILIC advances: experimental and theoretical progress clarified HILIC retention mechanisms, improved equilibration/gradient strategies and provided practical guidance for 2‑D coupling (HILIC×RPLC).
• Large molecules: Middle‑up HILIC‑HRMS and subunit analysis replaced laborious released‑glycan workflows for many glycoform quantitations; affinity CE‑MS and native SEC‑MS/AF4‑MS approaches enabled proteoform‑resolved binding and aggregation studies.
• Fast/ultra‑fast GC: Thermal‑gradient and flow‑field thermal gradient GC deliver major throughput gains when paired with TOFMS; retention stability and sample discrimination were mitigated by active cooling, purged connectors and optimized injector designs.
• Sample preparation and miniaturization: MIPs/IIPs and capillary monoliths enable selective extraction and on‑line workflows; automated DBS extraction coupled to LC‑MS/MS showed clinical‑grade performance for immunosuppressant TDM after hematocrit correction; 3D printed sorbents and cartridges proved useful for rapid prototyping and tailored SPE formats.
• Multidimensional separations and modulation theory: New insights into injection/loop dispersion, the "total breakthrough" phenomenon and models for GC×GC and LC×LC optimization were presented, improving method translation and 2D peak capacity.
• Data and machine learning: Graph neural networks and improved QSRR models substantially improved retention time predictability, aiding candidate identification and reducing false positives in untargeted workflows.

Benefits and practical applications

Hyphenated techniques validated at HTC‑17 translate into concrete benefits: faster QC and batch screening (fast GC, automated LC‑GC×GC), higher confidence identification for regulatory contexts (GC×GC‑TOFMS, high‑resolution MS, TIMS), improved biopharmaceutical characterization (middle‑up HILIC, affinity CE‑MS, SEC‑MS), better environmental monitoring (TD‑GC×GC, GC‑APCI‑TIMS‑MS), and portability of workflows (automated DBS, miniaturized CE‑MS). Advances in sample prep reduce matrix effects and increase sensitivity for low‑volume or trace analyses.

Future trends and opportunities

• Standardization and clinical translation: harmonized protocols, QC materials and multi‑site trials (e.g., Metabo‑Ring style) are needed to move breathomics and volatilomics into the clinic.
• Detector and interface innovation: LC detectors with improved structural specificity, and robust SFC‑MS and CE‑MS interfaces will expand hyphenation utility.
• More automation and continuous processing: on‑line MCSGP, automated LC–GC×GC platforms, and integrated sample‑prep/analysis pipelines will increase throughput while preserving data quality.
• Integration of ion mobility and mobility‑resolved MS: TIMS and other mobility stages will be used routinely for isomer and conformer resolution, especially for large molecules and complex environmental matrices.
• Machine learning and mechanistic modelling: graph neural networks for RT prediction, physics‑based models for band broadening, and automated interpretation of GC×GC datasets will accelerate method development and identification.
• Green and miniaturized chromatography: wider adoption of CO2‑based SFC, solvent reduction strategies, and 3D‑printed consumables to lower environmental footprint.
• Data FAIRness and workflow portability: shared special issues, community code, and vendor‑agnostic data pipelines will be important for reproducibility and regulatory acceptance.

Conclusion

HTC‑17 offered a concentrated snapshot of the field: hyphenation continues to expand both in depth (e.g., native proteoform analysis, TIMS, middle‑up glycoanalysis) and breadth (fast GC, unified SFC/LC modes, automated LC–GC×GC). Practical innovations in sample preparation, high‑throughput hardware and data science are closing the gap to routine industrial and clinical applications. Continued emphasis on standardization, robust interfaces and integrated informatics will be essential to fully translate these advances into regulated environments and large‑scale biological studies.

References

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• Schleich F.N., et al. Exhaled volatile organic compounds discriminate neutrophilic and eosinophilic asthma. American Journal of Respiratory and Critical Care Medicine 2019;200:444–453.
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